KR20210097750A - CD40 antibody pharmaceutical compositions and uses thereof - Google Patents

Abstract

The present invention provides a pharmaceutical composition comprising a CD40 antibody or antigen-binding fragment thereof, and an acetate-sodium acetate buffer solution, and uses thereof. The pharmaceutical composition may further comprise sugars, non-ionic surfactants and other excipients. The pharmaceutical composition of the present invention exhibits good antibody stability.

Description

CD40 antibody pharmaceutical compositions and uses thereof

This application claims priority to CN201811448238.5 (application date: November 30, 2018), the content of which is incorporated herein in its entirety.

The present application relates to the field of pharmaceutical formulations, and specifically to pharmaceutical compositions containing the CD40 antibody or antigen-binding fragment thereof, and to their use as medicaments.

As one of the glycoproteins expressed on the cell surface, CD40 is a type I membrane-internal glycoprotein with a molecular weight of 48 kDa, which belongs to the tumor necrosis factor receptor (TNFR) superfamily and plays an important role in the immune system. It is expressed in a variety of immune cells such as B cells, dendritic cells, monocytes and macrophages. When signal transduction is mediated by CD40, specialized antigen presenting cells will be activated. It is known that the natural ligand of CD40 is designated CD154 or CD40L and is predominantly expressed in mature T lymphocytes. CD40L-mediated signal transduction can trigger many cellular biological events, including activation and proliferation of immune cells, and production of cytokines and chemokines. CD40 signaling is extremely important in T cell-dependent immune responses, particularly in the tumor environment, where CD40-stimulated dendritic cells activate tumor-specific effector T cells, which have the potential to eradicate tumor cells.

Expression of CD40 has been found in a variety of normal cells and tumor cells, including B lymphocytes. For example, melanoma is one of the tumors that involve CD40 expression, and 30-70% of solid tumors also express CD40. Activation of CD40 is known to effectively trigger anti-tumor responses, including immune activation of tumor-specific T cell responses, direct apoptosis of CD40-positive tumors, and humoral responses of ADCC induced by stimulation. [Tong et al, Cancer Gene Therapy, 2003, 10: 1-13]). In addition, the observed tumor eradication is strongly associated with the presence of tumor-specific cytotoxic T lymphocytes. On the other hand, it should also not be disregarded that systemic administration of CD40 antibody is generally associated with various side effects, such as shock syndrome and cytokine release syndrome (van Mierlo et al, Proc. Nat1. Acad. Sci. USA, 2002, 99: 5561-5566]).

Currently, many global pharmaceutical companies are developing monoclonal antibodies against CD40 as described above, which specifically stimulate immune activation to maximize the patient's own immune system response to the tumor and thus achieve the goal of tumor cell death. . Relevant patents include: CN1198647, CN1369015, CN1582165, CN100430419, CN101014386, CN101237882, CN101289510, CN101490086, CN103842382, CN104918957, WO2002028904, WO2011123489, WO2011123489, WO2012149201700, WO20130324904, etc., WO196314, WO2015400091856, etc. So far, anti-CD40 antibodies from Pfizer (related products are licensed to Roche), Alligator and others have shown good tumor killing effects in preclinical animal models.

WO2018219327 (PCT/CN2018/089252) provides anti-CD40 antibodies with high affinity, high selectivity and high biological activity for use by stimulating CD40 antibodies.

However, antibody drugs with large molecular weights and complex structures can become unstable due to their tendency to degradation, polymerization, or experience of undesirable chemical alterations. In order to make the antibody suitable for administration and to maintain stability and better performance for storage and subsequent use, the study of stable formulations of antibody drugs is of great importance. An example of a patent related to a CD40 antibody formulation is WO2005063289. For new CD40 antibodies, there is still a need to develop pharmaceutical compositions (formulations) comprising CD40 that are more suitable for administration.

Provided herein are pharmaceutical compositions comprising a CD40 antibody or antigen-binding fragment thereof, and a buffer. In some embodiments, the buffer is selected from an acetate buffer, a histidine buffer, a phosphate buffer and a succinate buffer, preferably an acetate buffer, more preferably an acetate-sodium acetate buffer.

In an alternative embodiment, the CD40 antibody or antigen-binding fragment thereof of the pharmaceutical composition comprises LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively, and SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5 has HCDR1, HCDR2 and HCDR3.

In an alternative embodiment, the CD40 antibody or antigen-binding fragment thereof of the pharmaceutical composition has a concentration of about 1-120 mg/ml, which is about 1 mg/ml, 2 mg/ml, 3 mg/ml, 4 mg/ml. ml, 5 mg/ml, 6 mg/ml, 7 mg/ml, 8 mg/ml, 9 mg/ml, 10 mg/ml, 12 mg/ml, 14 mg/ml, 16 mg/ml, 18 mg/ml ml, 20 mg/ml, 22 mg/ml, 24 mg/ml, 26 mg/ml, 28 mg/ml, 30 mg/ml, 35 mg/ml, 40 mg/ml, 45 mg/ml, 50 mg/ml ml, 55 mg/ml, 60 mg/ml, 65 mg/ml, 70 mg/ml, 75 mg/ml, 80 mg/ml, 85 mg/ml, 90 mg/ml, 95 mg/ml, 100 mg/ml ml, 105 mg/ml, 110 mg/ml, 115 mg/ml, 120 mg/ml or any value between any two values, preferably 10-40 mg/ml, most preferably about 25 mg It can be /ml.

In an alternative embodiment, the buffer has a concentration of about 5 to 30 mM, preferably about 10 to 20 mM, non-limiting examples of which are about 10 mM, 12 mM, 14 mM, 16 mM, 18 mM, 20 mM. or any value between any two values, most preferably about 10 mM.

In an alternative embodiment, the buffer of the pharmaceutical composition has a pH of about 4.5 to 6.5, preferably about 5.0 to 6.0, and in non-limiting embodiments, it also has a pH of about 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0 or any value between any two values, most preferably about 5.0 or 5.5.

In an alternative embodiment, the pharmaceutical composition further comprises a sugar. "Sugar" herein includes conventional compositions (CH ₂ O) _n and derivatives thereof such as monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars and the like. As used herein, "sugar" means glucose, sucrose, trehalose, lactose, fructose, maltose, dextran, glycerin, erythritol, glycerol, arabitol, silitol, sorbitol, mannitol, melibiose, melezitose, raffinose, Mannotriose, stachyose, maltose, lactulose, maltulose, sorbitol, maltitol, lactitol, iso-maltulose, and the like. Preferred sugars are non-reducing disaccharides, more preferably trehalose and sucrose.

In an alternative embodiment, the sugar of the pharmaceutical composition has a concentration of about 40 to 95 mg/ml (eg 55 mg/ml), preferably about 60 to 90 mg/ml. In a non-limiting example, the concentration of sugar is 60 mg/ml, 65 mg/ml, 70 mg/ml, 75 mg/ml, 80 mg/ml, 85 mg/ml, 90 mg/ml or any two values between any value, more preferably about 80 mg/ml.

In an alternative embodiment, the pharmaceutical composition further comprises a surfactant, which comprises polysorbate 20; polysorbate 80; poloxamer; Triton; sodium dodecyl sulfonate; sodium lauryl sulfonate; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl-, stearyl-sarcosine; linoleyl-, myristyl-, cetyl-betaine; lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmamidopropyl-, isostearamidopropyl-betaine; myristamidopropyl-, palmamidopropyl-, isostearamidopropyl-dimethylamine; sodium methylcocoyl; sodium methyloleyl taurate; copolymers of polyethylene glycol; copolymers of polypropylene glycol; It may be selected from copolymers of ethylene glycol and copolymers of propylene glycol, and the like. Preferred surfactants are polysorbates, such as polysorbate 80 or polysorbate 20, more preferably polysorbate 80.

In an alternative embodiment, the surfactant in the pharmaceutical composition has a concentration of about 0.02 to 0.8 mg/ml (eg 0.1 mg/ml), preferably about 0.3 to 0.6 mg/ml. In a non-limiting example, the concentration of surfactant is 0.3 mg/ml, 0.35 mg/ml, 0.4 mg/ml, 0.45 mg/ml, 0.5 mg/ml, 0.55 mg/ml, 0.6 mg/ml or any two values. any value in between, more preferably about 0.4 mg/ml.

In an alternative embodiment, the pharmaceutical composition comprises:

(a) from 1 to 120 mg/ml of a CD40 antibody or antigen-binding fragment thereof, wherein the CD40 antibody or antigen-binding fragment thereof is LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively, and has HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, respectively), and

(b) 5-30 mM acetate buffer.

In an alternative embodiment, the pharmaceutical composition comprises, preferably the pharmaceutical composition has a pH of 4.5 to 6.5, more preferably 5.0 to 6.0, further preferably 5.5:

(a) from 1 to 120 mg/ml of a CD40 antibody or antigen-binding fragment thereof, wherein the CD40 antibody or antigen-binding fragment thereof is LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively, and has HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, respectively);

(b) 5 to 30 mM acetate buffer,

(c) 40 to 90 mg/ml of sucrose, and

(d) Polysorbate 80 at 0.02 to 0.8 mg/ml.

In an alternative embodiment, the pharmaceutical composition comprises, preferably the pharmaceutical composition has a pH of 4.5 to 6.5, more preferably 5.0 to 6.0, further preferably 5.5:

(a) from 1 to 120 mg/ml of a CD40 antibody or antigen-binding fragment thereof, wherein the CD40 antibody or antigen-binding fragment thereof is LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively, and has HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, respectively);

(b) 5 to 30 mM acetate buffer,

(c) 40 to 95 mg/ml of trehalose, and

(d) Polysorbate 80 at 0.02 to 0.8 mg/ml.

the pharmaceutical composition

(a) from 1 to 120 mg/ml of a CD40 antibody or antigen-binding fragment thereof, wherein the CD40 antibody or antigen-binding fragment thereof is LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively, and has HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, respectively), and

(b) 10-20 mM acetate buffer

Including, the pharmaceutical composition has a pH of 5.0 to 5.5.

In an alternative embodiment, the pharmaceutical composition comprises, preferably the pharmaceutical composition has a pH of 5.0 to 6.0:

(a) 10-40 mg/ml of a CD40 antibody or antigen-binding fragment thereof, wherein the CD40 antibody or antigen-binding fragment thereof is LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively, and has HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, respectively);

(b) 10-20 mM acetate buffer,

(c) 60 to 90 mg/ml of sucrose, and

(d) 0.3 to 0.8 mg/ml of polysorbate 80.

In an alternative embodiment, the pharmaceutical composition comprises, preferably the pharmaceutical composition has a pH of 5.0 to 6.0:

(a) 25 mg/ml of a CD40 antibody or antigen-binding fragment thereof, wherein the CD40 antibody or antigen-binding fragment thereof comprises LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively, and the sequence HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5),

(b) 10-20 mM acetate-sodium acetate buffer,

(c) 60 to 90 mg/ml of sucrose, and

(d) 0.3 to 0.8 mg/ml of polysorbate 80.

In a specific embodiment, the pharmaceutical composition comprises:

25 mg/ml CD40 antibody, 80 mg/ml sucrose, 0.6 mg/ml polysorbate 80 and 10 mM histidine-histidine hydrochloride, pH 5.5;

25 mg/ml CD40 antibody, 80 mg/ml sucrose, 0.6 mg/ml polysorbate 80 and 10 mM histidine-histidine hydrochloride, pH 5.8;

25 mg/ml CD40 antibody, 80 mg/ml sucrose, 0.6 mg/ml polysorbate 80 and 10 mM histidine-histidine hydrochloride, pH 6.0;

25 mg/ml CD40 antibody, 80 mg/ml sucrose, 0.4 mg/ml polysorbate 80 and 10 mM histidine-histidine hydrochloride, pH 5.6;

25 mg/ml CD40 antibody, 80 mg/ml sucrose, 0.4 mg/ml polysorbate 80 and 10 mM histidine-histidine hydrochloride, pH 5.8;

25 mg/ml CD40 antibody, 80 mg/ml sucrose, 0.4 mg/ml polysorbate 80 and 10 mM histidine-histidine hydrochloride, pH 6.0;

25 mg/ml CD40 antibody, 80 mg/ml sucrose, 0.4 mg/ml polysorbate 80 and 10 mM histidine-acetic acid buffer, pH 5.0;

25 mg/ml CD40 antibody, 80 mg/ml sucrose, 0.4 mg/ml polysorbate 80 and 10 mM histidine-acetic acid buffer, pH 5.5;

25 mg/ml CD40 antibody, 80 mg/ml sucrose, 0.4 mg/ml polysorbate 80 and 10 mM histidine-acetic acid buffer, pH 6.0;

25 mg/ml CD40 antibody, 80 mg/ml sucrose, 0.6 mg/ml polysorbate 80 and 10 mM histidine-acetic acid buffer, pH 5.0;

25 mg/ml CD40 antibody, 80 mg/ml sucrose, 0.6 mg/ml polysorbate 80 and 10 mM histidine-acetic acid buffer, pH 5.5;

25 mg/ml CD40 antibody, 80 mg/ml sucrose, 0.6 mg/ml polysorbate 80 and 10 mM histidine-acetic acid buffer, pH 5.8;

25 mg/ml CD40 antibody, 80 mg/ml sucrose, 0.6 mg/ml polysorbate 80, and 10 mM histidine-acetic acid buffer, pH 6.0;

25 mg/ml CD40 antibody, 80 mg/ml sucrose, 0.4 mg/ml polysorbate 80 and 10 mM acetate-sodium acetate buffer, pH 5.5; or

25 mg/ml CD40 antibody, 80 mg/ml sucrose, 0.6 mg/ml polysorbate 80 and 10 mM acetate-sodium acetate buffer, pH 5.5.

In the specific embodiment defined above, the CD40 antibody comprises LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively, and HCDR1, HCDR2 shown in SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, respectively and HCDR3.

In some embodiments, the CD40 antibody or antigen-binding fragment thereof herein has a heavy chain variable region shown in SEQ ID NO: 1 and a light chain variable region shown in SEQ ID NO: 2:

In an alternative embodiment, the antibody or antigen-binding fragment thereof of the pharmaceutical composition may be selected from a murine antibody, a chimeric antibody and a humanized antibody, preferably a humanized antibody.

In an alternative embodiment, the light chain FRs on the light chain variable region and the heavy chain FRs on the heavy chain variable region of the humanized antibody of the pharmaceutical composition are derived from human germline light and heavy chains, or mutant sequences thereof, respectively.

In addition, the heavy chain of the humanized CD40 antibody has the sequence shown in SEQ ID NO: 17 or a variant thereof, said variant preferably having 0 to 10 amino acid modifications in the heavy chain variable region, more preferably mutations at amino acid positions 6 and 8 provided that said amino acid is preferably mutated to I, A or L; The light chain of the humanized antibody has the sequence shown in SEQ ID NO: 18 or a variant thereof, wherein the variant preferably has 0 to 10 amino acid modifications in the light chain variable region, more preferably at amino acid positions 2 and 3, wherein Amino acids are preferably mutated to I, V or L.

In an alternative embodiment, the heavy chain variable region of the humanized CD40 antibody further comprises a heavy chain FR of a human IgG1, IgG2, IgG3 or IgG4 or a variant thereof, preferably comprising a heavy chain FR of a human IgG1, IgG2 or IgG4, , more preferably a heavy chain FR of human IgG1 or IgG2.

In an alternative embodiment, the amino acid sequence of the light chain of the CD40 antibody is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, wherein the amino acid sequence of the heavy chain of the antibody is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, wherein the sequence of the hu9E5 antibody light chain is It is shown in SEQ ID NO: 18, and the sequence of the hu9E5 antibody heavy chain is shown in SEQ ID NO: 17.

The pharmaceutical composition of the present application has sufficient drug stability and can be stored stably for a long period of time. On the other hand, for the convenience of drug transport, the pharmaceutical composition of the present application may be further produced as a lyophilized formulation.

Also provided herein is a method for producing a lyophilized formulation comprising a CD40 antibody, comprising the step of lyophilizing a pharmaceutical composition as defined above.

In an alternative embodiment, in a method for producing a formulation comprising a CD40 antibody, lyophilization comprises a pre-lyophilization step, a primary drying step, and a secondary drying step in this order.

Also provided herein is a lyophilized formulation comprising a CD40 antibody produced by a method for producing a lyophilized formulation comprising a CD40 antibody as defined above.

In some embodiments, the lyophilized formulation is stable at 2°C to 8°C for at least 3 months, at least 6 months, at least 12 months, at least 18 months, or at least 24 months.

In some embodiments, the lyophilized formulation is stable at 25°C for at least 3 months, at least 6 months, at least 12 months, at least 18 months, or at least 24 months.

In some embodiments, the lyophilized formulation is stable at 40° C. for at least 7 days, at least 14 days, or at least 28 days.

Also provided herein is a method for producing a reconstitution solution of a lyophilized formulation comprising a CD40 antibody, comprising reconstituting the lyophilized formulation as defined above, wherein the solution used for reconstitution comprises, but is not limited to, the main The use is selected from water, physiological saline or glucose solution.

Also provided herein is a reconstitution solution of a lyophilized formulation comprising a CD40 antibody produced by a method for producing a reconstitution solution of a lyophilized formulation containing a CD40 antibody as defined above.

Also provided herein are articles or kits comprising a container containing any stable pharmaceutical composition. In some embodiments, the vial is an injection vial made from a neutral borosilicate glass tube.

Also provided herein is the use of a pharmaceutical composition, a lyophilized formulation or a reconstituted solution of a lyophilized formulation in the production of a medicament for the treatment of a CD40-associated disease or condition, wherein the disease or condition is preferably cancer, The cancer is from lymphoma, breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, lung cancer, liver cancer, stomach cancer, colorectal cancer, bladder cancer, rhabdomyosarcoma, esophageal cancer, cervical cancer, multiple myeloma, leukemia, gallbladder cancer, glioblastoma and melanoma. is chosen

Also provided herein is a method for treating or preventing a CD40-associated disease or condition, comprising administering to a patient in need thereof a therapeutically effective amount of a pharmaceutical composition as defined herein, a lyophilized formulation or a reconstituted solution of the lyophilized formulation. administering, wherein the disease or condition is preferably cancer, wherein the cancer is lymphoma, breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, lung cancer, liver cancer, stomach cancer, colorectal cancer, bladder cancer, rhabdomyosarcoma , esophageal cancer, cervical cancer, multiple myeloma, leukemia, gallbladder cancer, glioblastoma and melanoma.

Also provided herein is a pharmaceutical composition as defined above, a lyophilized formulation or an article of manufacture comprising a container containing a reconstituted solution of the lyophilized formulation.

It should be understood that one, some, or all of the features of the various embodiments described herein may be combined to form other embodiments of the present disclosure. These and other aspects herein will be apparent to those skilled in the art. These and other embodiments herein are further described in the Detailed Description below.

1. Definition

In order to more readily understand the present disclosure to those skilled in the art, certain technical and scientific terms are specifically defined below. Unless expressly defined otherwise herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this application belongs.

"Buffer" refers to a buffer capable of resisting changes in pH through the action of an acid-base complex component. Examples of buffers capable of adjusting the pH in an appropriate range include acetate buffer, succinate buffer, gluconate buffer, histidine buffer, oxalate buffer, lactate buffer, phosphate buffer, citrate buffer, tartrate buffer, fumarate buffer, lysylglycine buffers and other organic acid buffers.

A “histidine buffer” is a buffer containing a histidine ion. Examples of the histidine buffer include a histidine-hydrochloride buffer, a histidine-acetate buffer, a histidine-phosphate buffer, a histidine-sulfate buffer, and the like, preferably a histidine-hydrochloride buffer. Histidine-hydrochloride buffers are prepared from histidine and hydrochloric acid or histidine and histidine hydrochloride.

A “citrate buffer” is a buffer containing citrate ions. Examples of the citrate buffer include citrate-sodium citrate buffer, citrate-potassium citrate, citrate-calcium citrate, citrate-magnesium citrate, and the like. A preferred citrate buffer is a citrate-sodium citrate buffer.

A “succinate buffer” is a buffer containing succinate ions. Examples of the succinate buffer include sodium succinate-succinate buffer, succinate-potassium succinate buffer, succinate-calcium succinate buffer, and the like. A preferred succinate buffer is a succinate-sodium succinate buffer.

A “phosphate buffer” is a buffer containing phosphate ions. Examples of the phosphate buffer include disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, disodium hydrogen phosphate-potassium dihydrogen phosphate buffer, and the like. A preferred phosphate buffer is a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer.

"Acetate buffer" is a buffer containing acetate ions. Examples of the acetate buffer include an acetate-sodium acetate buffer, an acetate-histidine buffer, an acetate-potassium acetate buffer, an acetate-calcium acetate buffer, an acetate-magnesium acetate buffer, and the like. A preferred acetate buffer is an acetate-sodium acetate buffer.

A "pharmaceutical composition" is a mixture containing one or more compounds described herein, or a physiologically/pharmaceutically acceptable salt or prodrug thereof, and other chemical ingredients, such as physiologically/pharmaceutically acceptable carriers and excipients. it means. The purpose of a pharmaceutical composition is to exercise its biological activity by facilitating administration to an organism and facilitating absorption of the active ingredient. In this context, "pharmaceutical composition" and "agent" are not mutually exclusive.

In solution forms of the pharmaceutical compositions described herein, unless otherwise specified, the solvent therein is water.

“Lyophilized formulation” refers to a formulation or pharmaceutical composition obtained by vacuum freeze-drying of a pharmaceutical composition or solution formulation in liquid or solution form.

As used herein, the term “about” means that an index value is within an acceptable error range of a specific value determined by one of ordinary skill in the art, and is dependent, in part, on how the value is determined or measured (ie, the limits of the measurement system). For example, “about” can mean one or more than one standard deviation in all implementations of the technique. Alternatively, “about” or “comprising substantially” may mean a range of 20% or less. Also, particularly for biological systems or processes, the term may mean up to 10 times or up to 5 times a value. Unless otherwise stated, when a specific value appears in the present application and in the claims, it should be assumed that the meaning of "about" or "comprising substantially" is within an acceptable error range of the specific value.

The pharmaceutical compositions described herein must achieve a stable effect: the antibody therein must substantially retain its physical and/or chemical stability and/or biological activity after storage. Preferably, the pharmaceutical composition retains its physical stability, chemical stability and biological activity after storage. The shelf life is generally selected based on a predetermined shelf life of the pharmaceutical composition. There are a variety of analytical techniques available to measure the stability of a protein, which can measure stability after storage at a selected temperature for a selected period of time.

A stable pharmaceutical antibody preparation is one in which no significant change is observed under the following conditions: at least 1 month, preferably 6 months, more preferably 1 year, even more preferably at refrigerated storage temperature (2°C to 8°C). Stored for up to 2 years. Stable liquid formulations also include liquid formulations that exhibit the desired characteristics after a period comprising storage at 25° C. for 1 month, 3 months or 6 months, or storage at 40° C. for 1 month. Typical acceptable criteria for stability are as follows: generally no more than about 10%, preferably no more than about 5% of the antibody monomers are degraded as measured by SEC-HPLC. As determined by visual analysis, the pharmaceutical antibody preparation is colorless or clear to slightly pale white. The concentration, pH and osmotic pressure of the formulations have deviations of ±10% or less. A cleavage of about 10% or less, preferably about 5%, is generally observed, and about 10% or less, preferably about 5% or less of agglomerates are generally formed.

If the antibody does not show a significant increase in aggregation, precipitation and/or denaturation after visual examination of color and/or clarity, or as measured by ultraviolet scattering, size exclusion chromatography (SEC) and dynamic light scattering (DLS), the antibody maintains its physical stability in pharmaceutical formulations. Changes in protein structure can be assessed by fluorescence spectroscopy (which determines the tertiary structure of the protein) and FTIR spectroscopy (which determines the secondary structure of the protein).

If the antibody does not show significant chemical changes, the antibody retains its chemical stability in pharmaceutical formulations. Chemical stability can be measured by detecting and quantifying a protein having a chemically modified form. Degradation processes that often change the chemical structure of proteins are hydrolysis or cleavage (assessed by methods such as size exclusion chromatography and SDS-PAGE), oxidation (peptide mapping in combination with methods such as mass spectrometry or MALDI/TOF/MS, etc.) ), deamidation (assessed by methods such as ion exchange chromatography, capillary isoelectric focusing, peptide mapping, isoaspartic acid measurement, etc.) and isomerization (assessed by isoaspartic acid content determination, peptide mapping, etc.) include

The antibody retains its biological activity in the pharmaceutical formulation if, at a given time, the biological activity of the antibody is within a predetermined range of the biological activity exhibited when the pharmaceutical formulation was prepared. The biological activity of an antibody can be determined, for example, by antigen binding assays.

The term “CD40” refers to a cell surface receptor that is a member of the TNF receptor superfamily, also known as TNFRSF5. CD40 is ubiquitously expressed on the surface of dendritic cells, B cells and macrophages, and is a molecule necessary for the production and maintenance of T cell immunity. The term “CD40” encompasses any variant or isoform of CD40 that is natively produced by a cell. The antibodies herein may be cross-reactive with CD40 of a non-human species. Alternatively, the antibody may be human CD40-specific and may not exhibit cross-reactivity with other species. CD40 or any variant or isoform thereof may be isolated from a cell or tissue that naturally expresses it or is produced by recombinant techniques using techniques conventional in the art and described herein. Preferably, the anti-CD40 antibody targets human CD40 with a normal glycosylation pattern.

As used herein, amino acid three-letter codes and one-letter codes are described in J. Biol. Chem, 243, p3558 (1968).

"Antibody" as referred to herein refers to an immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy and two light chains linked by an interchain disulfide bond.

As used herein, the antibody light chain may further comprise a light chain constant region comprising a human or murine κ or λ chain, or a variant thereof.

As used herein, the antibody heavy chain may further comprise a heavy chain constant region comprising a human or murine IgG1, IgG2, IgG3 or IgG4, or variant thereof.

Variable fragments (Fv regions) of about 110 amino acids close to the N-terminus of antibody heavy and light chains can differ significantly in their amino acid sequences. The constant region consisting of the remaining amino acid sequence close to the C-terminus of the antibody is relatively stable. The variable region comprises three hypervariable regions (HVRs) and four relatively conserved framework regions (FRs). The three hypervariable regions that determine the specificity of an antibody are also referred to as complementarity determining regions (CDRs). Each of the light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. the three CDRs of the light chain refer to LCDR1, LCDR2 and LCDR3; The three CDRs of the heavy chain refer to HCDR1, HCDR2 and HCDR3. The number and position of CDR amino acid residues of the LCVR and HCVR of the antibody or antigen-binding fragment herein conforms to the known Kabat numbering standards (LCDR1-3, HCDR2-3) or the Kabat and Chothia numbering system. (HCDR1) is compliant.

Antibodies herein include murine antibodies, chimeric antibodies and humanized antibodies, preferably humanized antibodies.

As used herein, the term “murine antibody” is a monoclonal antibody targeting human CD40 produced according to the knowledge and skill in the art. After injection of the CD40 antigen into the test subject during production, hybridomas expressing the antibody with the desired sequence or functional characteristics are isolated.

The term “chimeric antibody” is an antibody produced by fusing the variable region of a murine antibody with the constant region of a human antibody, which is capable of ameliorating the immune response induced by the murine antibody. To construct chimeric antibodies, hybridomas secreting murine-specific monoclonal antibodies are first constructed, and then variable region genes are cloned from murine hybridoma cells. Then, the constant region gene of the human antibody is cloned as needed. After ligation of the murine variable region gene and the human constant region gene into the chimeric gene, it is inserted into a human vector, and finally the chimeric antibody molecule is expressed in eukaryotic or prokaryotic industrial systems. In a preferred embodiment herein, the light chain of the chimeric CD40 antibody or antigen-binding fragment thereof may further comprise a light chain constant region of a human κ or λ chain or a variant thereof. The heavy chain of the chimeric CD40 antibody further comprises a heavy chain constant region of a human IgG1, IgG2, IgG3 or IgG4, or variant thereof.

Also, the term "humanized antibody" (also known as CDR-grafted antibody) refers to the grafting of murine CDR sequences into the framework of a human antibody variable region (i.e. the framework sequences of a different type of human germline antibody). It refers to the antibody produced by This can overcome the strong immune response induced by chimeric antibodies containing large amounts of murine protein components. Such framework sequences can be obtained from public DNA databases or published references that contain germline antibody gene sequences. For example, the germline DNA sequences of the variable region genes of human heavy and light chains are described in the "VBase" human germline sequence database (available at www.mrccpe.com.ac.uk/vbase), and in the literature [ Kabat, EA, etc., Human, 1991 Sequences of Proteins of Immunological Interest, 5th edition. To avoid a decrease in activity caused by reducing immunogenicity, human antibody variable regions can be minimally backmutated to maintain activity. The humanized antibodies herein also include humanized antibodies that have further undergone affinity maturation to CDRs by phage display.

The term “binding to CD40” herein refers to the ability to interact with human CD40.

The term "specific binding" is determined by techniques available in the art, such as competition ELISA, BIACORE® assay or KINEXA® assay. The term is also applicable, for example, when the antigen-binding domain of an antibody herein is specific for a particular epitope encompassed by many antigens, in which case an antibody having an antigen-binding domain binds to a plurality of antigens bearing the epitope. It can bind specifically.

"Antigen-binding fragments" herein are Fab fragments, Fab' fragments, F(ab')2 fragments and scFv fragments that bind to human CD40, and are produced by using the VH and VL of antibodies capable of binding to human CD40. Refers to other fragments capable of binding to human CD40. It comprises one or more CDRs of an antibody described herein selected from SEQ ID NOs: 7, 13, 9, 10, 11 and 12. An Fv fragment is the smallest antibody fragment comprising the heavy and light chain variable regions of an antibody, but no constant regions, and all antigen binding sites. Generally, Fv antibodies also contain a polypeptide linker between the VH and VL domains that can form the structure necessary for antigen binding. In addition, different linkers can be used to link the two variable regions of an antibody into a polypeptide chain, which is referred to as a single chain antibody or single chain Fv (sFv).

The term “epitope” refers to a portion of a molecule capable of recognizing an antibody by one or more antigen binding regions of the antibody and capable of binding by the antibody.

"Conservative modifications" or "conservative substitutions or replacements" refer to amino acids of a protein that have similar characteristics (e.g., charge, side chain size, hydrophobicity/hydrophilicity, main chain structure and stiffness, etc.) so that they can often be changed without altering the biological activity of the protein. It refers to substitution with another amino acid having It is generally known to those skilled in the art that single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter its biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/ Cummings Pub. Co., Page 224, (4th edition)]. In addition, substitution of structurally or functionally similar amino acids is unlikely to interfere with their biological activity.

"Identity" or "homology" of amino acid sequences refers to sequence similarity between two polypeptide sequences. Molecules are identical at that position, e.g., when the same amino acid residue occupies a position on both sides of two compared sequences, e.g., when the same amino acid occupies the same position in each of two polypeptides. Examples of suitable algorithms for determining percent sequence identity and percent sequence similarity are the BLAST and BLAST2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol. 215:403-410] and [Altschul et al. (1977) Nucleic Acids Res. 25:3389-3402]. Software for performing BLAST analysis is publicly available from the US National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/).

Methods for the production and purification of antibodies and antigen-binding fragments are well known in the prior art, such as Chapters 5-8 and 15, Using Antibodies: A Laboratory Manual published by Cold Spring Harbor. An antibody or antigen-binding fragment as defined herein is genetically engineered to add one or more human FRs to non-human CDRs. Human FR germline sequences were obtained by alignment with the IMGT human antibody variable region germline gene database and MOE software (ImMunoGeneTics (IMGT) website [http://imgt.cines.fr] or the Journal of Immunoglobulins, 2001ISBN012441351). can be obtained.

The engineered antibodies or antigen-binding fragments herein can be produced and purified by conventional methods. For example, cDNA sequences encoding the heavy and light chains can be cloned into a GS expression vector and recombined. CHO cells can be stably transfected with recombinant immunoglobulin expression vectors. As a more recommended art-well-known method, mammalian expression systems will result in glycosylation of the antibody, particularly at the highly conserved N-terminus of the Fc. Stable clones are obtained by expressing an antibody that specifically binds to a human antigen. Positive clones are expanded in a bioreactor in serum-free medium to produce antibodies. The culture medium in which the antibody is secreted can be purified and collected by incubation techniques. For example, an A or G Sepharose FF column with adjusted buffer is used for purification to wash off components that are not specifically bound. The bound antibody is then eluted by a pH gradient and the antibody fragments are collected after detection by SDS-PAGE. Antibodies can be concentrated by filtration in a conventional manner. In addition, soluble mixtures and multimers can be removed by conventional methods such as molecular sieves and ion exchange. The product produced should be immediately frozen (eg -70° C.) or lyophilized.

When applied to an animal, human, laboratory subject, cell, tissue, organ, or biological fluid, "administration" and "treatment" refer to exogenous drug, therapeutic agent, diagnostic agent or composition to the animal, human, laboratory subject, cell, tissue, organ. or contact with a biological fluid. "Administration" and "treatment" refer to, for example, therapeutic methods, pharmacokinetic methods, diagnostic methods, investigational methods, and laboratory methods. Treatment of a cell includes contacting a reagent to the cell, and contacting the reagent to a fluid, wherein the fluid is in contact with the cell. In addition, "administration" and "treatment" refer to, for example, treatment of cells in vitro and ex vivo with reagents, diagnostic agents or binding compositions or with another type of cell. “Treatment” refers to therapeutic treatment, prophylaxis or prevention means for research and diagnostic applications when applied to human, veterinary subjects or research subjects.

"Therapy" means administering to a patient having one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect, eg, any composition capable of binding a therapeutic agent, eg, a compound herein, for internal or external use. do. In general, a therapeutic agent is administered to induce a decrease in symptoms or inhibit the progression of such symptoms to any clinically unmeasurable extent in an amount that effectively alleviates the symptoms of one or more diseases in the individual or population to be treated. The amount of a therapeutic agent effective to alleviate the symptoms of any particular disease (also referred to as a "therapeutically effective amount") depends on a variety of factors, such as the stage of the disease, the age and weight of the patient, and the amount of the drug that induces the desired effect in the patient. It can change depending on your abilities. Whether the symptoms of the disease have been alleviated can be assessed by any clinical trial method by a physician or other health care professional to assess the severity or progression of the disease. Although embodiments herein (eg, methods of treatment and agents) may not be effective in ameliorating disease symptoms for their respective targets, any statistical testing method known in the art, such as Student's t-test, chi- Square test, U test based on Mann and Whitney, Kruskal-Wallis test (H test), Jonckheere-Terpstra test and Wilcoxon The target's disease symptoms can be alleviated in a statistically significant number of patients as determined by the assay.

An “effective amount” includes an amount sufficient to ameliorate or prevent a symptom or condition of a medical disease. Also, an effective amount means an amount sufficient to enable diagnosis. An effective amount for a particular patient or veterinary subject may vary depending on the following factors: for example, the disease to be treated, the patient's general health, the route and dosage of administration and the severity of side effects. An effective amount may be the maximum dosage or dosing regimen that avoids significant side effects or toxic effects.

"Tm value" refers to the temperature of protein thermal denaturation, ie, the temperature at which half of the protein is not folded when the spatial structure of the protein is disrupted. Therefore, the higher the Tm value, the higher the thermal stability of the protein.

2. Examples and test examples

The present application is further illustrated in detail by the following examples. These examples are for illustrative purposes only and do not limit the scope of the present application.

Experimental methods for which specific conditions are not indicated in the examples of the present application generally follow conventional conditions or conditions suggested by manufacturers or raw materials or raw materials. Reagents for which no specific source is indicated are commercially purchased conventional reagents.

Methods for the production and purification of the CD40 antigen and antibody herein are described in WO2018219327, the contents of which are incorporated herein in their entirety.

Example 1: Immune antigens, sequences of antigens for screening and production thereof

His-tagged human CD40 (h-CD40-his) recombinant protein, Fc-tagged human CD40 (h-CD40-Fc) recombinant protein, his-tagged murine CD40 (m-CD40-his) recombinant protein and his- Tagged rhesus CD40 (rhesus-CD40-his) recombinant protein (#CD0-C52H7) was a purified commercial protein reagent (purchased from Acrobiosystems). The data of each sequence is shown in Table 1 below. Proteins can be used in each of the experiments in the Examples below.

Example 2: Production of antibody hybridomas

Anti-human CD40 monoclonal antibody was immunized with mice (experimental C57BL/6 mice, females, 6-8 weeks old, Zhao Yan (Suzhou) New Drug Research Center Co., Ltd., Animal Production License No.: 201503259)). produced by Breeding environment: SPF level. Mice were purchased and bred in a laboratory environment at a temperature of 20° C. to 25° C. and a humidity of 40 to 60% in a 12/12 hour light/dark cycle control for one week. The acclimatized mice were divided into two cages, 5 in each cage.

The immune antigen was Fc-tagged modified human CD40 recombinant protein (h-CD40-Fc formulated in phosphate buffer at a concentration of 1 μg/μl). Freud's adjuvant (Sigma, lot number: F5881/F5506) was used for emulsification, where Freud's complete adjuvant (CFA) was used for primary immunization, nucleic acid adjuvant (CpG, Sangon Biotech) and aluminum for injection (Imject Alum) (Thermo, lot number: PH203866) was used for the remaining boost immunizations. Immunizations were performed on days 0, 14, 28, 42, 56 and 70. Blood was collected for blood tests on days 21, 35, 49, 63 and 77, and mouse serum was detected by ELISA to determine the antibody titer of the mouse serum.

After the fourth immunization, spleen cell fusions were performed in mice, where antibody titers tended to be high and stable in serum. Boost immunization was performed 3 days prior to fusion, and antigen solution (10 μg) formulated using phosphate buffer was injected intraperitoneally (IP) in each mouse. The splenic lymphocytes were fused with myeloma cells Sp2/0 cells (ATCC(R) CRL-8287TM) using an optimized PEG-mediated fusion step to obtain hybridoma cells, monoclonal hybrids with good in vitro activity. A hybridoma cell line was selected.

Example 3: Cloning and sequencing of anti-CD40 antibody

Hybridoma subclones of the antibodies screened and identified above were selected, and logarithmic growth stage hybridoma cells were collected. RNA was extracted and reverse transcribed using Trizol (Invitrogen, 15596-018) according to the kit's instructions (PrimeScript™ Reverse Transcriptase, Takara, catalog number: 2680A). The cDNA produced was then amplified by PCR using a murine Ig-primer set (Novagen, TB326 Rev. B 0503) and sent to a sequencing company for sequencing. The sequence of the murine antibody was finally obtained.

The heavy chain variable region and light chain variable region sequences of 9E5 are as follows:

The CDR sequences of 9E5 are shown in Table 2:

The obtained variable region sequence was grafted onto each of the constant region sequences of human antibody IgG1 to obtain a human-murine chimeric antibody sequence, and the sequence of the chimeric antibody was transferred to a pCP expression vector (purchased from MabSpace biology Co., Ltd.) by molecular cloning technique. ) was inserted into After these sequences were amplified by PCR (molecular biology working methods, such as molecular cloning, were carried out according to the usual operating conditions and can be specifically referred to "Molecular Cloning: A Laboratory Manual"), they were sequenced and identified. , human-murine chimeric antibody 9E5-C was obtained using the HEK293 cell expression system. Various in vitro activity assays were performed to characterize the purified chimeric antibodies by MabSelect SuRe (GE Lifesciences) affinity chromatography. The data are presented in Table 3 below.

Example 4: Humanization of Murine Antibodies

According to the typical VH/VL CDR structure of the obtained murine antibody 9E5, the heavy and light chain variable region sequences were compared with the antibody germline database to obtain a human germline template with high homology. Among them, the human germline light chain framework region was derived from a human κ light chain gene, and the light chain framework region of the antibody of the present invention was preferably a human germline light chain template Vk2-28/JK4(9E5). The human germline heavy chain framework region was derived from a human heavy chain, and the heavy chain framework region of the antibody of the invention was preferably a human germline heavy chain template VH1-2/JH6(9E5) as shown below:

The heavy chain framework region of 9E5 is preferably human germline heavy chain template IGHV1-2 (SEQ ID NO: 9):

The light chain framework region of 9E5 is preferably human germline light chain template IGkV2-28 (SEQ ID NO: 10):

The CDRs of the murine antibody were grafted onto selected humanized templates to replace the humanized variable regions, followed by recombination with the corresponding human IgG constant regions (preferably the heavy chain was IgG1 and the light chain was κ). Then, based on the three-dimensional structure of the murine antibody, the built-in residues, i.e. those that directly interact with the CDRs, and those that have important influence on the structure of VL and VH are backmutated, and chemically unstable amino acid residues of the CDRs are optimized. to obtain the final humanized molecule.

The final humanized hu9E5 antibody molecule (comprising the H1c heavy chain and the L1a light chain) was selected through the above expression test and comparison of the number of back mutations in the combination of light and heavy chains, of which the complete light and heavy chain sequences were SEQ ID NO: 17, respectively. and 18.

Example 5: Test Data of Humanized Antibodies

The binding and blocking activities of the humanized antibody hu9E5 of the present invention against human and rhesus CD40 are shown in Table 4 below.

Example 6: Inhibition of Anti-CD40 Antibodies on Tumor Growth in Mice

Normal human peripheral blood was collected and healthy human PBMCs were isolated by density gradient centrifugation. Monocytes were ^{sorted using the CD14 +} microbead kit, and CD14 ⁺ monocytes were isolated according to the protocol provided with the kit, i.e., 20 μl of anti-CD14 microbeads were ^{added for each of 10 7} cells and 15 at 4°C. incubated for minutes. The cells were then added to the magnetic column and rinsed 3 times. Cells from the magnetic column, ie CD14 ⁺ monocytes, were collected. Induction culture of MoDC cells by adding RPMI 1640 medium containing 10 ng/ml IL4 and 100 ng/ml GM-CSF to CD14 ⁺ monocytes by culturing the monocytes for 6 days (the culture method is a common method in the art) was performed. RPMI 1640 containing IL-2 was added to the remaining cells, and the suspended cells were collected after culturing (the culture method and the cell collection method are all routine methods in the art). T cells were sorted using a ^{CD3 + microbead kit.} After 6 days, MoDC cells and CD3 ⁺ T cells were each collected and mixed with Raji cells (Shanghai Institute of Biotechnology Cell Bank, cultured in RPMI1640 medium containing 10% fetal bovine serum) in a ratio of 1:5:20 and each of NOG mice (Nanjing Galaxy Biopharma, bred for acclimatization for 5 days) were subcutaneously inoculated. Experimental animals were housed in separate ventilated boxes with constant temperature and humidity. The temperature of the breeding room was 18.0° C. to 26.0° C., the humidity was 40 to 70%, and ventilation was performed at 10 to 20 times/hour, and the contrast was replaced with 12 hours/12 hours.

The experimental group included a human IgG1 antibody control group, hu9E5, and a reference antibody G12, all administered at a dose of 3 mg/kg. 5 mice in each group were dosed once a week for 6 weeks as 3 consecutive doses. During the entire experiment, the longitudinal and abscissa diameters of the tumors were measured twice a week using a vernier caliper, and the tumor volume was calculated as follows: tumor volume (mm ³ ) = 0.5 x (tumor longitudinal diameter x tumor abscissa) diameter ² ). The relative tumor inhibition ratio (TGI (%)) was calculated as follows: TGI% = (1-T/C) x 100%. % T/C is the relative tumor growth ratio, i.e., the percentage of the relative tumor volume or tumor weight of treated and control groups in a particular trial. T and C are the tumor volume (TV) or tumor weight (TW) of the treatment group and the IgG1 control group, respectively, at a specific time point.

The results indicated that the humanized anti-CD40 antibody hu9E5 had a very significant anti-tumor effect compared to the IgG1 control, with almost complete tumor clearance after 21 days of administration.

Exemplary Methods of Production of Antibody Pharmaceutical Compositions (Formulations)

First step: CD40 antibody (eg hu9E5) and stable formulation were produced in bulk formulation containing CD40 antibody.

After filtration, the bulk was sampled for sterility testing. The bulk was then filtered through a 0.22 μm PVDF filter element and the filtrate was collected.

Second step: The filling volume was adjusted to 1.1 ml and the filtrate was filled into a 2 ml vial equipped with a stopper. Samples were taken at the start, middle and end of fill to measure different fill volumes.

Step 3: Turn on the capping machine to perform capping with an aluminum cap.

Step 4: The product was confirmed free of defects such as incorrect filling by visual inspection. The vial label was then printed and pasted. Then, while the carton box was folded, the carton label was printed. The product was packaged. Finally, a box label was applied to the box.

Example 7: Screening of pH Values of Buffer Systems for Anti-CD40 Antibody Formulations

The following buffers were formulated to produce antibody preparations, where the anti-CD40 antibody concentration was 50 mg/ml, and samples were taken for stability studies at high temperature (40° C.).

1) 10 mM acetate-sodium acetate, pH 5.0;

2) 10 mM acetate-sodium acetate, pH 5.5;

3) 10 mM succinate-sodium succinate, pH 5.0;

4) 10 mM succinate-sodium succinate, pH 5.5;

5) 10 mM succinate-sodium succinate, pH 6.0;

6) 10 mM histidine-histidine hydrochloride, pH 5.5;

7) 10 mM histidine-histidine hydrochloride, pH 6.0;

8) 10 mM histidine-histidine hydrochloride, pH 6.5.

The results show that the optimal pH range for the anti-CD40 antibody is 5.0 to 6.0. In this pH range, the difference between the three buffer systems in each of SEC and CE is not significant, and the deviation of ICE is within the range of 14.0 to 24.1%, which is more stable than the buffer system (buffer) with pH 6.5.

Example 8: Screening of Surfactants for Anti-CD40 Antibody Formulations

A buffer system of 10 mM histidine-histidine hydrochloride (pH 5.5) and 10 mM acetate-sodium acetate (pH 5.5) was selected to contain 50 mg/ml protein, 75 mg/ml sucrose, and different types of surfactants. An anti-CD40 antibody formulation was produced. The stability of its appearance at 25° C. was investigated:

1) containing 10 mM histidine-histidine hydrochloride, pH 5.5, 0.4 mg/ml polysorbate 80;

2) containing 10 mM histidine-histidine hydrochloride, pH 5.5, 0.4 mg/ml polysorbate 20;

3) containing 10 mM acetate-sodium acetate, pH 5.5, 0.4 mg/ml polysorbate 80;

4) containing 10 mM acetate-sodium acetate, pH 5.5, 0.4 mg/ml polysorbate 20.

The results show that polysorbate 80 is the preferred surfactant.

Example 9: Screening of Surfactant Concentrations for Anti-CD40 Antibody Formulations

A buffer system with 10 mM acetate-sodium acetate pH 5.5 was selected to produce anti-CD40 antibody formulations containing 50 mg/ml protein, 75 mg/ml sucrose and different concentrations of polysorbate 80:

1) containing 10 mM acetate-sodium acetate, pH 5.5, 0.1 mg/ml polysorbate 80;

2) containing 10 mM acetate-sodium acetate, pH 5.5, 0.2 mg/ml polysorbate 80;

3) containing 10 mM acetate-sodium acetate, pH 5.5, 0.4 mg/ml polysorbate 80;

4) containing 10 mM acetate-sodium acetate, pH 5.5, 0.6 mg/ml polysorbate 80;

5) containing 10 mM acetate-sodium acetate, pH 5.5, 0.8 mg/ml polysorbate 80.

Samples were diluted to a protein concentration of 0.5 mg/ml each by injection of 0.9% sodium chloride. The results of the insoluble particles (see Table 3) show that the anti-CD40 antibody protein has good compatibility with sodium chloride when the concentration of polysorbate 80 is 0.1 mg/ml to 0.8 mg/ml, which is described above It is suggested that the polysorbate 80 concentration has a good stabilizing effect; Therefore, the samples described above were placed in a shaking incubator (25° C., 300 rpm) for 5 days. Depending on the appearance, the concentration of polysorbate 80 was chosen to be 0.4 to 0.8 mg/ml, preferably 0.4 mg/ml.

Example 10: Screening of Sugar Concentrations for Anti-CD40 Antibody Formulations

A buffer system with 10 mM acetate-sodium acetate pH 5.5 was selected to produce anti-CD40 antibody formulations containing 50 mg/ml protein, 0.4 mg/ml polysorbate 80 and various concentrations of sucrose:

1) containing 10 mM acetate-sodium acetate, pH 5.5, 55 mg/ml sucrose;

2) containing 10 mM acetate-sodium acetate, pH 5.5, 65 mg/ml sucrose;

3) containing 10 mM acetate-sodium acetate, pH 5.5, 70 mg/ml sucrose;

4) containing 10 mM acetate-sodium acetate, pH 5.5, 75 mg/ml sucrose;

5) 10 mM acetate-sodium acetate, pH 5.5, containing 80 mg/ml sucrose.

The osmolality results of each sample (see Table 8) show that the formulation is isotonic when the sucrose concentration is 80 mg/ml.

Example 11: Stability of Various Buffer Systems for Anti-CD40 Antibodies

The following buffer system was used to produce an anti-CD40 formulation containing 50 mg/ml protein, 80 mg/ml sucrose and 0.4 mg/ml polysorbate 80, and the stability was investigated at 25°C and 2°C to 8°C. did.

1) 10 mM acetate-sodium acetate, pH 5.5;

2) 10 mM histidine-histidine hydrochloride, pH 5.5;

3) 10 mM histidine-acetic acid, pH 5.5.

The results showed that after 6 months of storage at 25°C, the difference between 10 mM histidine-histidine hydrochloride and 10 mM histidine-acetic acid buffer systems was not significant, but the 10 mM acetate-sodium acetate buffer system was slightly superior to the rest of the buffer systems. ; After 6 months of storage at 2°C to 8°C, there was no significant difference between the quality of the three systems.

Example 12: Effect of protein concentration of formulation on stability

Select a buffer system with 10 mM histidine-hydrochloride (pH 5.5) to 80 mg/ml sucrose, 0.4 mg/ml polysorbate 80, and 50 mg/ml, 25 mg/ml or 1 mg/ml protein A CD40 antibody preparation containing the concentration was produced. High temperature stability at 40°C was investigated.

1) 10 mM histidine-histidine hydrochloride, pH 5.5, with a protein concentration of 50 mg/ml;

2) 10 mM histidine-histidine hydrochloride, pH 5.5, with a protein concentration of mg/ml;

3) 10 mM histidine-histidine hydrochloride, pH 5.5, with a protein concentration of 1 mg/ml.

The results show that the protein concentration of the formulation will affect the purity, the protein concentrations of 50 mg/ml and 25 mg/ml have little effect on the purity, and the stability of the formulation with the protein concentration of 1 mg/ml is clearly low shows

Example 13: Comprehensive Screening of Formulation Components

To further optimize protein concentration, polysorbate 80 concentration and pH, JMP software was used for DOE design, and RSM model was applied to obtain a set of rules (see below). Since there were no particularly significant differences between the stability data of the buffer systems histidine-histidine hydrochloride (His-HCl) and histidine-acetic acid (His-AA) and the pH buffer range of His-HCl was greater than 5.5, His-AA was replaced with DOE. It was chosen as a buffer for screening experiments. In a buffer system with 10 mM histidine-acetic acid (His-AA), anti-CD40 antibody formulations with various protein concentrations, various polysorbate 80 concentrations and 80 mg/ml sucrose were produced, and samples were used for stability analysis. Stored at 40°C:

(1) containing 0.4 mg/ml polysorbate 80, pH 5.0, 40 mg/ml CD40;

(2) containing 0.2 mg/ml polysorbate 80, pH 5.5, 10 mg/ml CD40;

(3) containing 0.4 mg/ml polysorbate 80, pH 5.5, 25 mg/ml CD40;

(4) containing 0.4 mg/ml polysorbate 80, pH 5.5, 25 mg/ml CD40;

(5) containing 0.4 mg/ml polysorbate 80, pH 5.0, 10 mg/ml CD40;

(6) containing 0.6 mg/ml polysorbate 80, pH 6.0, 40 mg/ml CD40;

(7) containing 0.2 mg/ml polysorbate 80, pH 5.0, 25 mg/ml CD40;

(8) containing 0.2 mg/ml polysorbate 80, pH 6.0, 40 mg/ml CD40;

(9) containing 0.6 mg/ml polysorbate 80, pH 5.0, 25 mg/ml CD40;

(10) containing 0.4 mg/ml polysorbate 80, pH 5.5, 40 mg/ml CD40;

(11) containing 0.6 mg/ml polysorbate 80, pH 5.5, 10 mg/ml CD40;

(12) containing 0.4 mg/ml polysorbate 80, pH 6.0, 10 mg/ml CD40.

The results show the following: when the pH value is 5.0 to 6.0, polysorbate 80 is 0.2 to 0.6 mg/ml, and the protein concentration is 10 to 40 mg/ml, the fluctuation of these parameters has a great influence on the stability did not reach The defined pH range was 5.0 to 6.0, so the median of 5.5 was taken as the final pH value, taking into account the previous screening results as a whole, 0.4 mg/ml was taken as the final concentration of polysorbate 80, and the protein concentration was taken as 25 mg/m ml.

Example 14: Lyophilization Stability of Anti-CD40 Antibodies in Various Buffer Systems

Because acetic acid (sodium acetate) buffer systems can shift pH values after reconstitution due to volatilization during lyophilization, they are not suitable for use as buffer systems for lyophilized formulations. 50 mg/ml protein, 80 mg/ml with a choice of two buffers, such as 10 mM histidine-histidine hydrochloride (His-HCl) (pH 5.5) and 10 mM histidine-acetic acid (His-AA) (pH 5.5) An anti-CD40 antibody formulation containing sucrose and 0.4 mg/ml polysorbate 80 was produced. The filling volume was 1.2 ml/bottle and the lyophilization process was as shown in Table 13:

1) 10 mM histidine-histidine hydrochloride, pH 5.5;

2) 10 mM histidine-acetic acid, pH 5.5.

The lyophilized product had a good cake shape, the appearance was clear after reconstitution, and no obvious changes in pH and purity were observed, indicating that the lyophilization process was appropriate. The results showed the following: Both kinds of buffers, such as 10 mM histidine-histidine hydrochloride (pH 5.5) and 10 mM histidine-acetic acid (pH 5.5), were suitable as buffer systems for the anti-CD40 antibody formulation.

Example 15: Other Alternative Agents

The present invention also provides other anti-CD40 antibody pharmaceutical formulations having different compositions including, but not limited to, other formulations including, but not limited to:

(1) 25 mg/ml CD40 antibody, 80 mg/ml sucrose, 0.6 mg/ml polysorbate 80 and 10 mM stidine-histidine hydrochloride, pH 5.5;

(2) 25 mg/ml CD40 antibody, 80 mg/ml sucrose, 0.6 mg/ml polysorbate 80 and 10 mM histidine-histidine hydrochloride, pH 5.8;

(3) 25 mg/ml CD40 antibody, 80 mg/ml sucrose, 0.6 mg/ml polysorbate 80 and 10 mM histidine-histidine hydrochloride, pH 6.0;

(4) 25 mg/ml CD40 antibody, 80 mg/ml sucrose, 0.4 mg/ml polysorbate 80 and 10 mM histidine-histidine hydrochloride, pH 5.6;

(5) 25 mg/ml CD40 antibody, 80 mg/ml sucrose, 0.4 mg/ml polysorbate 80 and 10 mM histidine-histidine hydrochloride, pH 5.8;

(6) 25 mg/ml CD40 antibody, 80 mg/ml sucrose, 0.4 mg/ml polysorbate 80 and 10 mM histidine-histidine hydrochloride, pH 6.0;

(7) 25 mg/ml CD40 antibody, 80 mg/ml sucrose, 0.4 mg/ml polysorbate 80 and 10 mM histidine-acetic acid buffer, pH 5.0;

(8) 25 mg/ml CD40 antibody, 80 mg/ml sucrose, 0.4 mg/ml polysorbate 80 and 10 mM histidine-acetic acid buffer, pH 5.5;

(9) 25 mg/ml CD40 antibody, 80 mg/ml sucrose, 0.4 mg/ml polysorbate 80 and 10 mM histidine-acetic acid buffer, pH 6.0;

(10) 25 mg/ml CD40 antibody, 80 mg/ml sucrose, 0.6 mg/ml polysorbate 80 and 10 mM histidine-acetic acid buffer, pH 5.0;

(11) 25 mg/ml CD40 antibody, 80 mg/ml sucrose, 0.6 mg/ml polysorbate 80 and 10 mM histidine-acetic acid buffer, pH 5.5;

(12) 25 mg/ml CD40 antibody, 80 mg/ml sucrose, 0.6 mg/ml polysorbate 80 and 10 mM histidine-acetic acid buffer, pH 5.8;

(13) 25 mg/ml CD40 antibody, 80 mg/ml sucrose, 0.6 mg/ml polysorbate 80 and 10 mM histidine-acetic acid buffer, pH 6.0;

(14) 25 mg/ml CD40 antibody, 80 mg/ml sucrose, 0.4 mg/ml polysorbate 80 and 10 mM acetate-sodium acetate buffer, pH 5.5;

(15) 25 mg/ml CD40 antibody, 80 mg/ml sucrose, 0.6 mg/ml polysorbate 80 and 10 mM acetate-sodium acetate buffer, pH 5.5.

The experimental results show that the CD40 antibody formulation with the formulation defined above has good stability and can be applied to the production of CD40 antibody medicaments.

SEQUENCE LISTING <110> JIANGSU HENGRUI MEDICINE CO., LTD. SHANGHAI HENGRUI PHARMACEUTICAL CO., LTD. <120> CD40 ANTIBODY PHARMACEUTICAL COMPOSITION AND USE THEREOF <130> P21412628KR <140> PCT/CN2019/121985 <141> 2019-11-29 <150> CN201811448238.5 <151> 2018-11-30 <160> 18 <170> PatentIn version 3.5 <210> 1 <211> 112 <212> PRT <213> Mus musculus <400> 1 Gln Val Gln Leu Gln Gln Pro Gly Ala Asp Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Ile Leu Thr Thr Tyr 20 25 30 Trp Ile Thr Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asp Ile His Pro Gly Ser Gly Ser Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Ser Lys Ala Thr Leu Thr Val Asp Thr Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Thr Arg Leu Ser Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Asp Tyr Trp Gly Gin Gly Thr Thr Leu Thr Val Ser Ser 100 105 110 <210> 2 <211> 112 <212> PRT <213> Mus musculus <400> 2 Asp Val Leu Met Thr Gln Ser Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Asn Ile Val Asn Ser 20 25 30 Gln Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Glu Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Thr Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Ala 85 90 95 Ser Leu Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 3 <211> 10 <212> PRT <213> Mus musculus <400> 3 Gly Tyr Ile Leu Thr Thr Tyr Trp Ile Thr 1 5 10 <210> 4 <211> 17 <212> PRT <213> Mus musculus <400> 4 Asp Ile His Pro Gly Ser Gly Ser Thr Lys Tyr Asn Glu Lys Phe Lys 1 5 10 15 Ser <210> 5 <211> 3 <212> PRT <213> Mus musculus <400> 5 000 <210> 6 <211> 16 <212> PRT <213> Mus musculus <400> 6 Arg Ser Ser Gln Asn Ile Val Asn Ser Gln Gly Asn Thr Tyr Leu Glu 1 5 10 15 <210> 7 <211> 7 <212> PRT <213> (Mus musculus) <400> 7 Lys Val Thr Asn Arg Phe Ser 1 5 <210> 8 <211> 9 <212> PRT <213> Mus musculus <400> 8 Phe Gln Ala Ser Leu Val Pro Trp Thr 1 5 <210> 9 <211> 98 <212> PRT <213> Artificial Sequence <220> <223> human germline heavy chain template IGHV1-2 <400> 9 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg <210> 10 <211> 100 <212> PRT <213> Artificial Sequence <220> <223> human germline light chain template IGkV2-28 <400> 10 Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser 20 25 30 Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala 85 90 95 Leu Gln Thr Pro 100 <210> 11 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> hu9E5-H1a <400> 11 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr 20 25 30 Trp Ile Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Asp Ile His Pro Gly Ser Gly Ser Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Ser Arg Val Thr Met Thr Val Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Asp Tyr Trp Gly Gly Gly Thr Thr Val Thr Val Ser Ser 100 105 110 <210> 12 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> hu9E5-H1b <400> 12 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr 20 25 30 Trp Ile Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Asp Ile His Pro Gly Ser Gly Ser Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Ser Arg Val Thr Leu Thr Val Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Asp Tyr Trp Gly Gly Gly Thr Thr Val Thr Val Ser Ser 100 105 110 <210> 13 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> hu9E5-H1c <400> 13 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ile Leu Thr Thr Tyr 20 25 30 Trp Ile Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Asp Ile His Pro Gly Ser Gly Ser Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Ser Arg Val Thr Leu Thr Val Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Asp Tyr Trp Gly Gly Gly Thr Thr Val Thr Val Ser Ser 100 105 110 <210> 14 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> hu9E5-L1a <400> 14 Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Asn Ile Val Asn Ser 20 25 30 Gln Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Lys Val Thr Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Ala 85 90 95 Ser Leu Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 15 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> hu9E5-L1b <400> 15 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Asn Ile Val Asn Ser 20 25 30 Gln Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Lys Val Thr Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Ala 85 90 95 Ser Leu Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 16 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> hu9E5-L1c <400> 16 Asp Val Leu Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Asn Ile Val Asn Ser 20 25 30 Gln Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Lys Val Thr Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Ala 85 90 95 Ser Leu Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 17 <211> 442 <212> PRT <213> Artificial Sequence <220> <223> hu9E5 HC <400> 17 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ile Leu Thr Thr Tyr 20 25 30 Trp Ile Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Asp Ile His Pro Gly Ser Gly Ser Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Ser Arg Val Thr Leu Thr Val Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Asp Tyr Trp Gly Gly Gly Thr Thr Val Thr Val Ser Ser 100 105 110 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 115 120 125 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 130 135 140 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 145 150 155 160 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 165 170 175 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 180 185 190 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 195 200 205 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 210 215 220 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 225 230 235 240 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 245 250 255 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 260 265 270 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 275 280 285 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 290 295 300 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 305 310 315 320 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 325 330 335 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 340 345 350 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 355 360 365 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 370 375 380 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 385 390 395 400 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 405 410 415 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 420 425 430 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 <210> 18 <211> 219 <212> PRT <213> Artificial Sequence <220> <223> hu9E5 LC <400> 18 Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Asn Ile Val Asn Ser 20 25 30 Gln Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Lys Val Thr Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Ala 85 90 95 Ser Leu Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 145 150 155 160 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215

Claims (20)

A pharmaceutical composition comprising a CD40 antibody or antigen-binding fragment thereof, and a buffer, wherein said CD40 antibody or antigen-binding fragment thereof comprises LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively, and HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5; wherein said buffer is preferably selected from acetate buffers, histidine buffers, phosphate buffers and succinate buffers, more preferably acetate buffers, most preferably acetate-sodium acetate buffers. According to claim 1,
A pharmaceutical composition, wherein the CD40 antibody or antigen-binding fragment thereof has a concentration of 1 to 120 mg/ml, preferably 10 to 40 mg/ml. 3. The method of claim 1 or 2,
A pharmaceutical composition having a pH of 4.5 to 6.5, preferably 5.0 to 6.0. 4. The method according to any one of claims 1 to 3,
A pharmaceutical composition, wherein the buffer has a concentration of 5 to 30 mM, preferably 10 to 20 mM. 5. The method according to any one of claims 1 to 4,
A pharmaceutical composition further comprising a disaccharide selected from trehalose and sucrose. 6. The method of claim 5,
A pharmaceutical composition, wherein the disaccharide has a concentration of 40 to 95 mg/ml, preferably 60 to 90 mg/ml. 7. The method according to any one of claims 1 to 6,
A pharmaceutical composition further comprising a surfactant which is polysorbate, more preferably polysorbate 80. 8. The method of claim 7,
A pharmaceutical composition, wherein the surfactant has a concentration of 0.02 to 0.8 mg/ml, preferably 0.3 to 0.8 mg/ml. (a) 1-120 mg having LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively, and HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, respectively /ml of CD40 antibody or antigen-binding fragment thereof,
(b) 5 to 30 mM acetate buffer,
(c) 40 to 90 mg/ml of sucrose, and
(d) 0.02 to 0.8 mg/ml of polysorbate 80
As a pharmaceutical composition comprising a,
Preferably, the pharmaceutical composition has a pH of 4.5 to 6.5, more preferably 5.0 to 6.0, further preferably 5.5. (a) 1-120 mg having LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively, and HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, respectively /ml of a CD40 antibody or antigen-binding fragment thereof, and
(b) 10-20 mM acetate buffer
As a pharmaceutical composition comprising a,
A pharmaceutical composition having a pH of 5.0 to 5.5. 11. The method according to any one of claims 1 to 10,
A pharmaceutical composition, wherein the CD40 antibody or antigen-binding fragment thereof has a heavy chain variable region shown in SEQ ID NO: 1 and a light chain variable region shown in SEQ ID NO: 2. 12. The method of claim 11,
A pharmaceutical composition, wherein the CD40 antibody or antigen-binding fragment thereof is a humanized antibody. 13. The method of claim 12,
A pharmaceutical composition, wherein the light chain FR sequence on the light chain variable region and the heavy chain FR sequence on the heavy chain variable region of the humanized antibody are derived from human germline light and heavy chains, or mutant sequences thereof, respectively. 14. The method of claim 12 or 13,
wherein the heavy chain of the humanized antibody has the sequence shown in SEQ ID NO: 17 or a variant thereof, said variant preferably having 0 to 10 amino acid modifications in the heavy chain variable region, more preferably mutations at amino acid positions 6 and 8, wherein the amino acid is preferably mutated to I, A or L;
wherein the light chain of the humanized antibody has the sequence shown in SEQ ID NO: 18 or a variant thereof, wherein the variant preferably has 0 to 10 amino acid modifications of the light chain variable region, more preferably mutations at amino acid positions 2 and 3, wherein The pharmaceutical composition, wherein the amino acid is preferably mutated to I, V or L. 15. The method according to any one of claims 12 to 14,
The heavy chain variable region of the humanized antibody further comprises a heavy chain FR of human IgG1, IgG2, IgG3 or IgG4 or a variant thereof, preferably comprises a heavy chain FR of human IgG1, IgG2 or IgG4, more preferably human IgG1 or a heavy chain FR of IgG2. 11. The method according to any one of claims 1 to 10,
A pharmaceutical composition, wherein the amino acid sequence of the light chain of the CD40 antibody has at least 90% sequence identity with the sequence shown in SEQ ID NO: 18, and the amino acid sequence of the heavy chain of the CD40 antibody has at least 90% sequence identity with the sequence shown in SEQ ID NO: 17. A method for producing a lyophilized formulation comprising a CD40 antibody, the method comprising:
17. A method comprising the step of lyophilizing the pharmaceutical composition of any one of claims 1 to 16. A lyophilized formulation comprising the CD40 antibody produced by the method of claim 17 . 19. Use of the pharmaceutical composition of any one of claims 1 to 16 or the lyophilized formulation of claim 18 in the production of a medicament for the treatment of a CD40-associated disease or condition, wherein the disease or condition is preferably cancer; The cancer is selected from lymphoma, breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, lung cancer, liver cancer, stomach cancer, colorectal cancer, bladder cancer, rhabdomyosarcoma, esophageal cancer, cervical cancer, multiple myeloma, leukemia, gallbladder cancer, glioblastoma and melanoma to be used. A product comprising a container, wherein the container contains the pharmaceutical composition of any one of claims 1 to 16 or the lyophilized formulation of claim 18 .